Amylase chemically coupled to cellulose ethers

ABSTRACT

A water insoluble amylase comprising Alpha - Beta - or gamma -amylase chemically coupled to p-diazophenoxy hydroxypropyl cellulose or Alpha - or Beta - amylase chemically coupled to pisothiocyanatophenoxy hydroxypropyl cellulose. These are prepared by dissolving Alpha -, Beta - or gamma -amylase in a buffer within a pH range of 6.3-7.7 and reacting at 0*-5* C. with the pdiazophenoxy hydroxypropyl ether of cellulose or by dissolving Alpha - or Beta -amylase in a buffer at a pH of approximately 8.6 with a p-isothiocyanato phenoxy hydroxypropyl ether of cellulose.

United States Patent Inventors Appl. No

Priority Sidney Alan Barker;

Peter John Somers, both of Birmingham; Roger Epton, Wolverhampton, all of England Oct. 16, 1969 Dec. 14, 1971 Ranks Boris McDougall Limited London, England Oct. 23, 1968 Great Britain AMYLASE CHEMICALLY COUPLED T0 CELLULOSE ETHERS 18 Claims, No Drawings U.S. Cl 195/63, l95/DlG. ll, l95/68 Int. Cl C07g 7/02 Field oi Search 195/63, 63

le s-W- References Cited OTHER REFERENCES Manecke; Von 0., Die Naturwissenschaften Vol. 5i p. 25- 33, 1964.

Axen et aL, Nature Vol. 2 10, p. 367- 369, i966. Silman et al., Annual Review of Biochemistry Vol. 35, part ii, p. 873- 883, i966.

Primary ExaminerLionel M. Shapiro Assistant Examiner-Gary M. Nath Attorney-Stevens, Davis, Miller & Mosher ing aor B-amylase in a buffer at a pH of approximately 8.6

with a p-isothiocyanato phenoxy hydroxypropyl ether of cellu- AMYLASE CHEMICALLY COUPLED T CELLULOSE ETHERS This invention is for improvements in or relating to enzymes and has particular reference to the modification of enzymes by attachment to solid matrices.

More particularly this invention relates to the water insolubilization of enzymes by chemically attaching them to cellulose derivatives and has as an object the provision of enzyme preparations in a form where they can be reused repeatedly and be more stable to heat than the corresponding soluble en zyme.

Water insoluble derivatives of a-amylase (diastase) have been prepared by chemical coupling of the enzyme to (a) nitrated copolymers of methacrylic acid, methacrylic acid mfluoranilide and divinyl benzene (G. Manecke and G. Gunzel, Maltromolekulare Chem., 51, (1962), 199 & G. Manecke, Pure Appl. Chem., 4 (1962), 507), and (b) nitrated copolymers of methacrylic acid, 4- or 3-fluorostyrene and divinyl benzene (G. Manecke and H. J. Forster Makromolekulare Chem., 91 (1966), l36). The enzyme activity of the bound protein in these preparations did not exceed 3 percent of that of the free enzyme in aqueous solution. Also the stability of these preparations was reported to be of only the same order as aqueous solutions of a-amylase stored under similar conditions.

It is well-known that when an enzyme is attached to an insoluble support the novel microenvironment of the enzyme markedly affects its stability. Hydrophilic features of the carrier tend to enhance the stability of the attached enzyme whereas hydrophobic features have the opposite effect. Polysaccharide carriers such as fibrous cellulose (M. A. Mitz and L. J. Summaria, Nature, London 189, (1961), 576 & W. E. Hornby, M. D., Lilly and E. M. Crook, Biochem, J., 98, (1966), 420) and cross-linked dextran (R. Axen and J. Porath, Nature, London 210, (1966), 367) have been shown to be particularly effective in conferring stability to the attached enzyme.

Commercial samples of water insoluble forms of trypsin, chymotrypsin, ribonuclease, glucose oxidase and ficin became available in Feb. i968. These were obtained by reaction of the appropriate enzyme with carboxymethyl cellulose hydrazide and are marketed by Seravac Laboratories, Ltd., Maidenhead, Berks.

Almost simultaneously (Jan. 1968) Miles Laboratories, lnc., Elkhart, U.S.A., marketed water insoluble forms of trypsin, chymotrypsin and papain in which the enzymes were bound to ethylene-maleic anhydride copolymer carrier.

R. Axen and .l. Porath, Nature 210 (1966), 367, succeeded in preparing active water insoluble chymotrypsin and trypsin by reaction of the enzymes with p-isothiocyanato phenoxy hydroxypropyl Sephadex (cross-linked dextran). They were unsuccessful in their attempt to obtain an active water insoluble B-amylase by the same process.

It is an object of the present invention to provide active water insoluble preparations of a-amylase, B-amylase and yamylase wherein the enzyme is chemically coupled with the pdiazophenoxy hydroxypropyl and p-isothiocyanato phenoxy hydroxy propyl ethers derived from microcrystalline cellulose.

The present invention provides a water insoluble amylase chemically coupled to p-diazophenoxy hydroxy propylor pisothiocyanato phenoxy hydroxypropyl cellulose.

More specifically the invention provides a, B- and yamylase chemically coupled to p-diazophenoxy hydroxy propyl cellulose and aand B-amylase chemically coupled to p-isothiocyanato phenoxy hydroxypropyl cellulose.

Water insoluble preparations of a-, ,6- or 'y-amylase may be made by chemical reaction at 05 C. of the amylase dissolved in a buffer within a pH range of 6.3-7.7 (preferably 7.6-7.7) with the p-diazophenoxyhydroxy propyl ether of cellulose. Unreacted diazo groups in the cellulose derivative are annealed by reaction with either B-naphthol or phenol. Preferably microcrystalline cellulose is used for the preparation of this ether and the degree of substitution of ether groups in the cellulose can be 13-562 microequivalents (preferably 13 microequivalents) of p-diazophenoxyhydroxypropyl ether groups per gram of cellulose. Active water insoluble preparations of a-amylase, B-amylase, glucamylase (y-amylase) can be obtained by this process which are more heat stable when suspended in an aqueous buffer (0.02 M) than the corresponding soluble enzyme. Preferably the buffer should have that pH at which the enzyme displays maximum enzymic activity towards its substrate.

Alternatively, water insoluble preparations of aor B- amylase may be made by chemical reaction at 0-S C. of aor B-amylase dissolved in a buffer (preferably 0.05 M borate bufier, pH 8.6) with the p-isothiocyanato-phenoxy-hydroxypropyl ether of cellulose. Preferably microcrystalline cellulose is used for the preparation of this ether and the degree of substitution of ether groups in the cellulose can be 13-562 microequivalents (preferably 13 microequivalents) of pisothiocyanato-phenoxy-hydroxypropyl ether groups per gram of cellulose. Active water insoluble preparations of a-amylase and B-amylase but not glucamylase (y-amylase) can be obtained by this method which are more heat stable when suspended in an aqueous buffer (0.02 M) than the corresponding soluble enzyme. Preferably the buffer should have that pH at which the enzyme displays maximum enzymic activity towards its substrate.

The particular merits of the present invention for providing water insoluble enzymes is that it can provide a product with a high retention of activity when calculated as a percentage of the activity which that amount of enzyme protein bound to the cellulose derivative would display in its original soluble form. The second advantage is that the process of the invention may be particularly advantageous for exoenzymes such as B- and 7- amylase (compare the water insoluble preparations of B- amylase obtained by reaction with the p-isothio-cyanato phenoxy hydroxypropyl ether of cross-linked dextran- Sephadex which were enzymically inactive, Axen and Porath, Nature, London 210 (1966) 367). The third advantage is that the use of microcrystalline cellulose in the preparation of the ether affords a dense hydrophilic carrier available in a fine particulate fonn for maximum surface exposure yet easily recoverable after use by centrifugation or filtration. The fourth advantage is the much greater heat stability of the water insoluble enzymes which may be obtained by the process of the present invention compared with the corresponding soluble enzyme giving a greater shelf life, a greater retention of activity at operating temperatures and enabling maximum repetitive use to be made of the enzyme.

Following is a description by way of example of methods of carrying the invention into effect.

EXAMPLE I p-Nitrophenol (69.5 g., 0.5 mole) was suspended in a solution of sodium hydroxide (25 g., 0.6 mole) in water (400 ml.) and the mixture heated to 70 C. to effect solution. On cooling slowly to room temperature with vigorous stirring finely divided sodium p-nitrophenoxide separated. Epichlorohydrin (46 g., 0.5 mole) was added and the solution gently stirred for 6 days at room temperature after which the solid was collected and washed on a filter with distilled water until free of bright yellow sodium p-nitro-phenoxide. The off-white damp solid was dissolved directly in ether (500 ml.) and the water layer which separated discarded. The ether layer was dried with magnesium sulfate and concentrated when a very pale yellow solid crystallized. Recrystallization from light petroleum (40/60) gave pure p-nitrophenylglycidyl ether 25.0 g. (25.5%), m.p. 67C., (E. Marle, J. Chem. Soc., 1912) 305).

Samples of microcrystalline cellulose (10 g., Sigmacel type 38 purchased from the Sigma Chemical Company, England) were placed in four SO-ml. stoppered round-bottomed flasks which were subsequently evacuated, left for 1 hour and filled with nitrogen. After warming each flask to 50 C. prewarmed aliquots (20 ml.) of a 10 percent solution of p-nitrophenylglycidyl ether were a dded followed by aliquots (10 ml.) of 10 percent aqueous sodium hydroxide solution. The contents of the flasks were then well mixed and stoppered. All operations were performed under nitrogen. The flasks were then mainmagnetically stirred for 15 minutes. The washing cycle was repeated three times. After the final washings had been decanted, aliquots (l ml.) of a 0.5 percent a-amylase solution (crystalline, ex Bacillus subtilis purchased from Sigma Chemitained at 50 C. After times of 12, 24, 48 and 96 hours the 5 cal Company) in phosphate butter (0.075 M, ph 7.6-7.7) rea tion mixture was tran ferred to a mortar and ground were added and the tubes stirred magnetically at -5 C. for lightly with 2N acetic acid before being suspended and stirred 18 hours when aliquots (5 ml.) of an ice-cold solution of B- for 0.5 hours with the same solvent (0.5L). The pnaphthol (0.! percent) in saturated sodium acetate was nitrophenoxy-hydroxy-propyl cellulose ethers were then coladded. After a further 15 minutes the water-insoluble o:- lected, washed and ground lightly with acetone and then amylase derivatives were subjected to five cycles of washing stirred with acetone (0.5L) for 0.5 hours. After three with Phosphate 1 PB and Sodium washings with distilled water (i L.) and one further washing chloride Solution 15 In the Same The with acetone (0.5 l...) the pale yellow ethers were collected on amylase derivatives were finally washed twice with P p a filter and n l5 bPf sii .k W... .1

RESULTS Percent activity t eq. active Mg. bound Enzyme retained functional protein/ units ling. by enzyme Functional oup active group/g. 100 mg. bound alter Prep. in protein b ding cellulose derivative protein coupling No.

p-Dlazophenoxyhydroxy- 0 04 0 6O 1 PH 232:; i123 iii? 33 i 56. 2 1. 66 33.3 37 4 1 One rat-amylase unit is that which liberates reducing sugar equivalent to 1 mg. maltose in 3 minutes at 0.

The degree of etherification was determined by titration of the p-amino-phenoxy-hydroxypropyl cellulose hydrochlorides with aqueous sodium hydroxide solution.

Degree of Substitution of p-Arninophcnoxy- Hydroxypropyl Cellulose Reaction time of p-nilrophcnylglycidyl ether with cellulose (h) peq p-amino-phenoxyhydroxypropyl ether per g. of cellulose EXAMPLE 11 Samples (100 mg.) of the four cellulose derivatives prepared in example 1 were placed in stoppered test tubes together with aliquots (5 ml.) of hydrochloric acid (I N). The tubes were placed in an ice bath and magnetically stirred for 15 minutes when aliquots (4 ml.) of ice-cold sodium nitrite solution were added. After a further 15 minutes the tubes were centrifuged, the supernatant discarded, aliquots (15 ml.) of ice-cold phosphate buffer (0.02 M, pH 6.3-6.4) added and the tubes magnetically stirred for 15 minutes. The washing cycle was repeated three times. After the final washings had been decanted, aliquots (l ml.) of a 0.5 percent a-amylase solution (crystalline, ex Bacillus subtil'is, purchased from Sigma Chemical Company) in phosphate buffer (0.02 M, pH 6.3-6.4) were added and the tubes stirred magnetically at 0-5' C. for 18 hours, when aliquots (5 ml.) of an ice-cold solution of B- naphthol (0.0] percent) in saturated sodium acetate were added. After a further 15 minutes the waterinsoluble aamylase derivatives were subjected to five cycles of washing with phosphate buffer (0.02 M, pH 6.9, 15 ml.) and sodium chloride (0.5 M) solution l5 ml.) in the same buffer. The aarnylase derivatives were finally washed twice with phosphate memo- 41 1 One tat-amylase unit is that which liberates reducing sugar equivalent to 1 mg. maltose in 3 minutes at 20 0.

Samples (I00 mg.) of the four cellulose derivatives prepared above were placed in stoppered test tubes together with aliquots (5 ml.) of hydrochloric acid (1 N). The tubes were placed in an ice bath and magnetically stirred for 15 minutes when aliquots (4 ml.) of ice-cold sodium nitrite solution were added. After a further l5 minutes the tubes were centrifuged, the supernatant discarded, aliquots (l5 ml.) of ice-cold phosphate buffer (0.075 M, pH 7.6-7.7) added and the tubes EXAMPLE iii Samples mg.) of the four p-amino-phenoxy-hydroxypropyl cellulose ethers prepared in example i were magnetically stirred into a slurry with aliquots (0.5 ml.) of phosphate buffer (3.5 M, pH 6.8). Aliquots (0.2 ml.) of i0 percent thiophosgene solution in carbon tetrachloride were added and stirring continued for 20 minutes, when further aliquots (0.2 ml.) of thiophosgene solution were added. After a further 20 minutes the p-isothiocyanato-phenoxy-hydroxypropyl cellulose ethers were washed once with acetone (15 ml.), twice with sodium bicarbonate solution (0.5 M, 15 ml.) and twice with borate bufler (0.05 M, pH 8.5, 15 ml.). After decantation of the final washings aliquots (1 ml.) of a 0.5 percent aamylase solution (crystalline, ex Bacillus subtilis, Sigma Chemical Company) in borate buffer (0.05 M, pH 8.5) were added and the tubes stirred magnetically at 0-5 C. for 18 hours. The a-amylase derivatives were subjected to five cycles of washing with phosphate buffer (0.02 M, pH 6.9, 15 ml.) and sodium chloride (0.5 M, 15 ml.) in the same buffer. The a-amylase derivatives were finally washed twice with phosphate buffer (0.02 M, pH 6.9).

buffer. After two further washings with acetate buffer (0.02 M, pH 4.5, ml.) and final decantation of the washings the yamylase derivative was resuspended in the same buffer (10 One amylase unit is taken as that which liberated reducing sugar equivalent to 1 mg. maltose at C. (B-amylase) or 1 mg. glucose at 45 C. ('ramylase) in 3minutes.

1 One a-amylase unit is that which liberates reducing sugar equivalent to 1 mg. maltose in 3 minutes at 20 0.

EXAMPLE 1V p-Amino-phenoxy-hydroxypropyl cellulose hydrochloride (100 mg., 20.6 ueq. ether linkage/g), prepared as in example I, was placed in a stoppered test tube and magnetically stirred into a slurry with phosphate buffer (3.5 M, pH 6.8, 0.5 ml.). Thiophosgene solution (10 percent, 0.2 ml.) in carbon tetrachloride was added and stirring continued for 20 minutes when a further aliquot (0.2 ml.) of thiophosgene solution was added. After stirring for a further 20 minutes acetone 15 ml.) was added and the solid p-isothiocyanatophenoxy-hydroxypropyl cellulose recovered by centrifugation. The washing cycle was repeated twice with sodium bicarbonate solution (0.5 M, 15 ml.) and twice with borate buffer (0.05 M, pH 8.6, 15 ml.). After decantation of the final washings a solution of B-amylase (crystalline, ex sweet potato, Sigma Chemical Company; 5 mg.) in borate buffer (0.05 M, pH 8.6, 1 ml.) was added and coupling allowed to proceed with gentle magnetic stirring for 48 hours at 0-5 C. The water-insoluble B-amylase derivative was subjected to five cycles of alternate washing with acetate buffer (0.02 M, pH 4.8, 15 ml.) and a solution of sodium chloride (1.0 M, 15 ml.) in the same buffer. After two further washings with acetate bufier (15 ml.) and final decantation of the washings the B-amylase derivative was resuspended in the same buffer 10 ml.).

EXAMPLE V p-lsothiocyanato-phenoxy-hydroxypropyl cellulose, prepared as above, was twice washed with sodium bicarbonate solution (0.5 M, 15 ml.) and twice with borate buffer (0.05 M, pH 8.6, 15 ml.). After decantation of the final washings a solution of y-amylase (glucamylase, ex Aspergillus Niger, partially purified by the starch procedure described by Cameron in Proc. Ciba Foundation Symposium .1. and A. Churchill Press, London 1967, 177; 5 mg. protein) in borate buffer (0.05 M, pH 8.6, 1 ml.) was added and coupling allowed to proceed with magnetic stirring for 48 hours at 05 C. The water-insoluble -y-amylase derivative was subjected to five cycles of alternate washing with acetate buffer (0.02 M, pH 4.5, 15 ml.) and a solution of sodium chloride (1.0 M, 15 ml.) in the same EXAMPLE Vl p-Amino-phenoxy-hydroxypropyl cellulose hydrochloride mg. 20.7 ueq. ether linkage/g.) prepared as described in example I, was stirred magnetically at 0 C. with hydrochloric acid (IN, 5 ml.). Sodium nitrite solution (2 percent, 4 ml.) precooled to 0 C., was added and stirring continued for 15 minutes. The p-diazo-phenoxy-hydroxypropyl cellulose was washed four times with phosphate buffer (0.075 M, pH 7.6-7.7, 15 ml.) at 0 C. After decantation of the final washings a solution of B-amylase (crystalline, ex sweet potato, purchased from Sigma Chemical Company, 5 mg.) in phosphate buffer (0.075 M, pH 7.6-7.7, 1 ml.) was added and coupling allowed to proceed with gentle magnetic stirring for 48 hours at 0-5 C. A solution of phenol (0.01 percent, 5 ml.) in saturated aqueous sodium acetate at 0 C. was then added. After a further 15 minutes stirring the water-insoluble B- amylase derivative was recovered by centrifugation.

Afier discarding the supernatant, the derivative was subjected to the washing procedure as described for the B- amylase derivative prepared by isothiocyanato coupling (example IV) and suspended in acetate buffer (0.02 M, pH 4.8, 10 ml.

EXAMPLE Vll p-Diazo-phenoxy-hydroxypropyl cellulose, prepared as above, was washed four times with phosphate buffer (0.075 M, pH 7.6-7.7, 15 ml.) at 0 C. After decantation of the final washings a solution of y-amylase (ex Aspergillus Niger, partially purified, 5 mg. protein) in phosphate buffer (0.075 M, pH 7.6-7.7, 1 ml.) was added and coupling allowed to proceed with gentle magnetic stirring for 48 hours at 0-5 C. A solution of phenol (0.01 percent. 5 ml.) in saturated sodium acetate at 0 C. was then added. After a further 15 minutes stirring the water insoluble y-amylase derivative was recovered by centrifugation. After discarding the supernatant, the derivative was subjected to the washing procedure described for the -y-amylase derivative prepared by isothiocyanato coupling (example V) and suspended in acetate buffer (0.02 M, pH 4.5, 10 ml.).

1 One amylase unit was taken as that which liberated reducing sugar equivalent to 1 mg.

maltose at 211 U. (fl-amylase) or 1 111g. glucose at 45 U. (7-a111y1u8e) 1n 6 minutes.

nee

EXAMPLE Vlll A sample of water-insoluble a-amylase (preparation 2, 20 mg.) was suspended in phosphate buffer (0.02 M, pH 6.9, 2 ml.) and incubated at 45 C. Aliquots (0.2 ml.) were withdrawn at times 0, 0.125, 1, 2, 4 and 7 days and pipetted directly into a magnetically stirred starch solution (10 ml. 1 percent) in phosphate buffer (0.02 M, pH 6.9) at 45 C. The activity of the water-insoluble a-amylase sample was then determined by periodic sampling and assay of the digests with dinitrosalicylate reagent according to Bernfeld [Methods in Enzymology (1955) 149]. Hence the percentage of the original activity remaining in the water-insoluble tit-amylase preparation was determined.

A control incubation was performed in which the water-insoluble wamylase was replaced by a solution of an equivalent amount of free a-amylase in phosphate bufi'er (0.02 M, pH 6.9, 2 ml.).

A sample of the diazo-coupled water-insoluble B-amylase suspension (preparation was maintained at 40 C. over 7 days and its activity determined at intervals against magnetically stirred starch solution as described above. A control incubation was performed in which the water-insoluble 3- amylase suspension was replaced by a solution of free [3 amylase in acetate bufi'er (0.02 M, pH 4.8). The percentage of the original activities remaining after various time intervals was then calculated. The experiment was repeated at 50 C. over an incubation period of 4 days.

The stability of 'y-amylase derivative (preparation 16) was determined by means of similar experiments performed over an incubation period of 4 days at 50 C. and 60 C. and over an incubation periodot' 2 days at 70 C.

Retention of activity on storage of water-insoluble 5- and y-amylase derivatives Preparation of l,2-Epoxy-3-(p-nitrophenoxy) propane (glycidyl p-nitrophenyl ether) p-Nitrophenol (695 g. 5 mole) was suspended in a solution of sodium hydroxide (250 g. 6 mole) in water (4 liters) and the mixture heated to 70 C. to effect solution. On cooling slowly to room temperature with vigorous stirring finely divided sodium p-nitrophenoxide separated. Epichlorohydrin (460 g. 5 mole) was added and the solution agitated in a 5-liter stainless steel vessel at room temperature after which the solid was collected and washed in a rolling ball-mill (160 r.p.m.) with distilled water until free of bright yellow sodium pnitrophenoxide. The off-white damp solid was dissolved directly in ethanol (400 ml.), and concentrated, a very pale yellow solid crystallizing (350 g.).

Preparation of 2-hydroxy-3-(p-nitrophenoxy) propyl ethers and 3-(p-amino-phenoxy )-2-hydroxypropyl ether "ufl s h r s s q l l s- EXAMPLE 1X Suspensions (l0 mgJml.) of water-insoluble a-amylase (preparations Nos. 1, 2, 3 and 4) in phosphate buffer (0.02 M, pH 6.9) were stored for 4 months at 05 C. The activity of the preparations was then determined, and the percentage of the original activity remaining calculated.

Retention of activity on storage of water-insoluble aemylase derivatives lb of original Prep. Mode of activity remaining Enzyme No. coupling after 4 months I Diazo- 66 2 62 a-amylase 3 7'3 4 '71 A sample of microcrystalline cellulose g.) Sigmacell type 38 purchased from Sigma Chemical Co., England] was placed in a 1 liter stoppered round-bottomed flask, evacuated and filled with nitrogen. After warming each flask to 50 C., 300 ml. of prewarmed solution of p-nitro'phenyl-glycidyl ether (as prepared above, 10 percent in toluene was added followed by 150 ml. of aqueous sodium hydroxide (10 percent). The contents of the flask were well mixed and stoppered. All operations were performed under nitrogen. The flasks were maintained at 50 C. After 48 hours the reaction mixture was transferred to a ball-mill and ground for 10 minutes at rpm. with acetic acid (2 N) before being suspended and stirred with the same solvent (1 liter). The p-nitro-phenoxyhydroxypropyl cellulose ether (2-hydroxy-3-( p-nitrophenoxy) propyl ether) was collected, washed with acetone, and then stirred with acetone for 0.5 hour. After three washings with distilled water and one further with acetone, the pale yellow ethers were collected and dried.

Reduction of the p-nitro-phenoxy-hydroxypropyl cellulose was effected by suspending 120 g. in a 1:1 solution of titanous chloride (12.5 percent) in hydrochloric acid (6N, 2.400 ml.) at 100 C. for 10 minutes. The p-amino-phenoxy-hydroxypropyl cellulose hydrochlorides (3-(p-aminophenoxy)-2- hydroxypropyl ether hydrochloride of cellulose) were collected on a filter and washed with hydrochloric acid (2N) until ree chassist tar qss brislsalihe a p wa washed three IOION 0510 times with distilled water and finally with acetone before being collected on a filter and dried.

Preparation of water-insoluble 'y-amylase derivatives by coupling y-amylase with diazotized 3-(p-amino-phenoxy)-2- hydroxypropyl ethers of cellulose Samples (25 g.) of the cellulose derivative prepared above were placed in beakers together with aliquots (1.25 liters) of hydrochloric acid (2 N). The beakers were placed in an ice bath and stirred for 15 minutes, when aliquots (1 liter) of icecold sodium nitrite solution (2 percent) were added. After a further 15 minutes the contents were centrifuged, the supernatant discarded, aliquots (1 liter) of ice-cold phosphate butTer (0.075 M, pH 7.6-7.7) added and the contents stirred for 15 minutes. The washing cycle was repeated three times. After the final washing aliquots (200 ml.) of solutions of commercial 'y-amylase preparations were added.

Group A-Dialysed enzyme, activity 35.5 units/mg. protein (Agidex, Glaxo). Group B-As above but activity 66.1 units/mg. protein. Group CNondialysed commercial enzyme (Novo), activity 44.2 units/mg. protein In each group solutions of varying protein concentrations were made up in acetate buffer (0.075 M, pH 4.5) to give applied protein concentrations in the range 420-4000 mg. protein/200 ml. The suspensions were stirred magnetically at -5 C. for 48 hours when aliquots 1.25 liters) of an ice-cold solution of B-naphthol (0.01 percent) in sodium acetate percent) were added. After a further minutes, the water insoluble 'y-amylase derivatives were subjected to five cycles of washing with acetate buffer (0.02 M, pH 4.5) (1 liter) and sodium chloride solution l M, 1 liter) in the same bufi'er. The y-amylase derivatives were finally washed twice with acetate buffer (0.02 M, pH 4.5).

One 'y-amylase unit liberates reducing sugars equivalent to p. mole of glucose in 1 minute at 45 C.

EXAMPLE XI p-Diazo-phenoxy-hydroxypropyl cellulose (100 mg.) prepared as in example 1 was washed four times with phosphate buffer (0.075 M, pH 4.5, 15 ml.) at 0 C. After decantation of the final washings a solution of 'y-amylase (ex. Aspergillus Niger, commercial preparation, 0.04 ml.) in acetate buffer (0.075 M, pH 4.5, 1 ml.) was added and coupling allowed to proceed with gentle stirring for periods from 12 to 72 hours. The addition of B-naphthol and washings were then carried as described in example X.

RESULTS Time of Activity Enzyme Bound protein Prepn. coupling units/mg. bound concentration No. hours protein mg./g. solid EXAMPLE Xll p-Diazo-phenyoxy-hydroxypropyl cellulose mg.) prepared as in example X was washed four times with phosphate buffer (0.075 M, pH 4.5, 15 ml.) at 0 C. After decantation of the final washings a solution of y-amylase (ex. A. Niger, crude commercial preparation, 0.04 ml.) in (a) phosphate bufi'er (0.075 M, pH 7.6, l ml.), (b) acetate buffer (0.075 M, pH 4.5, 1 ml.) was added and coupling allowed to proceed with gentle stirring. The addition of B-naphthol and washings were then carried out as described in example X.

RESULTS Activity Enzyme Prepn. Buffer units/mg. bound Bound protein No. solution protein mg./g. solid 30 Phosphate pH 7.6 2.3 29.8 31 Acetate pH 4.5 3.2 22.9

EXAMPLE Xlll As example XI, using a 48 hour coupling except for the reduction stage:

Reduction of the p-nitro-phenoxy-hydroxypropyl cellulose was effected by suspending l g. in citrate buffer (pH 5,8. 0.65 M, 30 ml.) with 25 ml. of titanous chloride solution (12.5 percent) and stirring at room temperature for 48 hours.

Samples of water insoluble y-amylase (total activity l00 units, 25 mg. protein/g. derivative) suspended in acetate bufier (0.02 M, pH 4.5, 10 ml.) were added to a stirred temperature controlled vessel (60 C.) containing maltose solu- .tions in acetate buffer (0.005 M, to 0.5 M, 250 ml.) and the initial velocity of the reaction producing glucose measured by following the glucose concentrations using the method of .Dalqvist, Biochem. J., 80, (1961) 547 to determine the glucose.

to suspensions of wheat-starch (10, 20, 30 percent) in acetate buffer (280 ml.) in a jacketed (60 C.) stirred vessel.

it. 12 RESULTS RESULTS initial reaction Starch Initial reaction initial maltose velocity mg. 5 Applied enzyme concn. rate mg. reducing Run N)v cmwm slucoselml'lhour Run No. activity w.lv.% sugurlmlJhour a 0.005 1.9 P 2: b 0.01 2.1 c on I 12 to 1 155 so as d 0.02 3.3

= 0.05 4.4 i iv* A 7 iWA A f 0.1 5.1 i EXAMPLE xvn i Suspensions containing 0.43 g. insoluble 'y-amylase (preparation 22) in acetate buffer (0.02 M, pH 4.5, ml.) A were applied to suspensions of wheat-starch which had been previously acid hydrolized to a dextrose equivalent (D.E.) of i2 percent, neutralized and dissolved in acetate buffer (0.02 EXAMPLE Xv 20 M, pH 4.5, 280 ml.) at 60C.

Suspensions of water insoluble y-amylase (preparation 22, 10.5 units/mg. bound protein, 36.9 mg. protein/g. of deriva- RESULTS tive) in acetate buffer (0.02 M, pH 4.5) were applied in various concentrations to suspensions of wheat-starch (20 percent 25 Initial tarch Initial reaction w./v.) in acetate buffer (300 ml.) in a jacketed (60 C.) stirred we g? vessel. The production of reducing sugars was followed by the Run hydrolysis) w./v,% sugarlmL/hour 96% hours method of ferricyanide reduction (Hagedorn-Jenson) and the results obtained from the slope of the curve at zero time. 5 m 40 6 r 20 so 24 u so 52 is RESULTS Applied enzyme concentration Initial reaction. EXAMPLE Xv!" Bound protein Activity rate mg. reduclng sufla'lmll'imuh Suspensions of enzyme preparations of aand B-amylase (Nos. 3, l5, 1 l and 13) were centrifuged down and the super- 1 8 84 s natant discarded. An aliquot (5ml.) of starch solution (1 per- 16 9 40 cent) in the appropriate buffer was added, the digest magneti- 333 cally stirred briefly and centrifuged down. A sample (1 ml.) 2: 2:: :3 was then pipetted into dinitrosalicylate reagent 1 ml.) as a u 79 830 42 zero time blank. The digest was magnetically stirred for IS minutes, centrifuged and part of the supernatant (1 ml.) W 45 pipetted into dinitrosalicylate reagent (1 ml.). Each remaining su ematant was discarded and each re aration stirred briefly XVI wii h 5 ml. of the same buffer that WBPS used before except that Suspensions containing 1.5 g. insoluble -y-amylase (preparano starch was present. After centrifuging down and discarding tion 17, 8.8 units/mg. protein, 12.6 mg. protein/g. of derivathe supernatant the experiment was repeated eight times in all. tive) in acetate buffer (0.02 M, pH 4.5, 20 ml.) were applied The supernatant from the final digest was further incubated at 20 C. no further digestion was apparent except in the case s eri ise 1 RESU'LTS Prep.N 0. Enzyme Mode of coupling Buffer Diazo coupled 0.02 M phosphate, pH 6.9 tat-Amylase: 3 A0 .D. 520 mn 1. 90 1. 04 0. 92 0. 87 0.77 0. 71 0. 70 0. 68 Timesused..-- 1 2 3 4 5 6 7 8 Percent activity remaining 48. 5 46 40. 5 37 37 36 Disco coupled 0.02 M acetate, pH 4.8

fl-Amylase: 15 AO.D.520 nm. 0.98 0.53 0.44 0. 37 0.32 0.27 0.24 0.22 Timesused 1 2 3 4 5 6 7 8 Percent activity remaining 54 45 38 32. 5 27. 5 24. 5 22. 5

Isothiocyanato 0.02 M phosphate, pH 6.9

(at-Amylase: u A0.D. 520 nm 1. 1. 22 I. 12 1. 01 0. 03 0.86 1. 81 0. 76 Timesused. 1 2 3 4 5 6 7 8 Percent activity remaining 73 07 61 56 52 49 46 Isothlocyanato 0.02 M acetate, pH 4.8

B-Amylssez a AOuD.520nm.. 1.40 0.78 0.82 0.54 0.50 0.45 0.40 0.30 Timesused 1 2 3 4 5 6 7 8 52 42 36 33. 5 30 27 24 Percent activity remaining What we claim is:

amylase which process comprises reacting at -5 C. a, B or 'y-amylase dissolved in a buffer within a pH range of 6.3 -7.7 with the p-diazophenoxy hydroxypropyl ether of microcrystalline cellulose.

2. A process as claimed in claim 1 wherein the pH range is between 7.6 and 7.7.

3. A process as claimed in claim 2 wherein unreacted diazo groups in the cellulose derivative are annealed by reaction with B-naphthol or phenol.

4. A process as claimed in claim 2 wherein the degree of substitution of ether groups in the cellulose is between microequivalents of p-isothiocyanato-phenoxy-hydroxypropyl ether groups per gram of cellulose.

8. A process as claimed in claim 1 wherein the pdiazophenoxy-hydroxypropyl ether of cellulose is prepared from 3-(p-aminophenoxy)-2-hydroxypropyl ether hydrochloride of cellulose by treatment with acidified sodium nitrite solutioniustprior to use for this purpose.

9. A process as claimed i; claim 8 wherein the 3-. aminophenoxy)-2-hydroxypropyl ether hydrochloride of cellulose is prepared by reducing 2-hydroxy-3-(p-nitrophenoxy) propyl ether of cellulose using titanous chloride in the presence of acid.

10. A process as claimed in claim 9 wherein a citrate buffer is employed in place of the acid.

11. A process as claimed in claim 9 wherein the Z-hydroxy- 3-(p-nitrophenoxy) propyl ether of cellulose is prepared by reaction of cellulose with glycidyl p-nitrophenyl ether under nitrogen in the presence of alkali.

12. A process as claimed in claim 9 wherein the 3-(paminophenoxy)-2-hydroxypropyl ether of cellulose is converted to 2-hydroxy-3-(p-isothiocyanatophenoxy) propyl ether of cellulose by treatment with thiophosgene in carbon tetrachloride.

l3. aAmylase chemically coupled to p-diazophenoxy hydroxypropyl cellulose.

14. B-Amylase chemically coupled to p-diazophenoxy hydroxypropyl cellulose.

15. 'y-Amylase chemically coupled to p-diazophenoxy hydroxypropyl cellulose.

16. a-Amylase chemically coupled to p-isothiocyanato phenoxy hydroxypropyl cellulose.

17. B-Amylase chemically coupled to p-isothiocyanato phenoxy hydroxypropyl cellulose.

18. A water insoluble amylase comprising a- B- or yamylase chemically coupled to p-diazophenoxy hydroxypropyl cellulose or aor B-amylase chemically coupled to pisothiocyanatophenoxy hydroxypropyl cellulose. 

2. A process as claimed in claim 1 wherein the pH range is between 7.6 and 7.7.
 3. A process as claimed in claim 2 wherein unreacted diazo groups in the cellulose derivative are annealed by reaction with Beta -naphthol or phenol.
 4. A process as claimed in claim 2 wherein the degree of substitution of ether groups in the cellulose is between 13.-56.2 microequivalents of p-diazophenoxy hydroxypropyl etheR groups per gram of cellulose.
 5. A process for the preparation of an active water insoluble amylase which process comprises reacting at 0*-5* C., Alpha or Beta -amylase dissolved in a buffer at a pH of approximately 8.6 with the P-isothiocyanato-phenoxy-hydroxypropyl ether of microcrystalline cellulose.
 6. A process as claimed in claim 5 wherein the buffer is 0.05 M borate buffer, pH 8.6.
 7. A process as claimed in claim 5 wherein the degree of substitution of ether groups in the cellulose is 13-56.2 microequivalents of p-isothiocyanato-phenoxy-hydroxypropyl ether groups per gram of cellulose.
 8. A process as claimed in claim 1 wherein the p-diazophenoxy-hydroxypropyl ether of cellulose is prepared from 3-(p-aminophenoxy)-2-hydroxypropyl ether hydrochloride of cellulose by treatment with acidified sodium nitrite solution just prior to use for this purpose.
 9. A process as claimed in claim 8 wherein the 3-(p-aminophenoxy)-2-hydroxypropyl ether hydrochloride of cellulose is prepared by reducing 2-hydroxy-3-(p-nitrophenoxy) propyl ether of cellulose using titanous chloride in the presence of acid.
 10. A process as claimed in claim 9 wherein a citrate buffer is employed in place of the acid.
 11. A process as claimed in claim 9 wherein the 2-hydroxy-3-(p-nitrophenoxy) propyl ether of cellulose is prepared by reaction of cellulose with glycidyl p-nitrophenyl ether under nitrogen in the presence of alkali.
 12. A process as claimed in claim 9 wherein the 3-(p-aminophenoxy)-2-hydroxypropyl ether of cellulose is converted to 2-hydroxy-3-(p-isothiocyanatophenoxy) propyl ether of cellulose by treatment with thiophosgene in carbon tetrachloride.
 13. Alpha -Amylase chemically coupled to p-diazophenoxy hydroxypropyl cellulose.
 14. Beta -Amylase chemically coupled to p-diazophenoxy hydroxypropyl cellulose.
 15. gamma -Amylase chemically coupled to p-diazophenoxy hydroxypropyl cellulose.
 16. Alpha -Amylase chemically coupled to p-isothiocyanato phenoxy hydroxypropyl cellulose.
 17. Beta -Amylase chemically coupled to p-isothiocyanato phenoxy hydroxypropyl cellulose.
 18. A water insoluble amylase comprising Alpha - Beta - or gamma -amylase chemically coupled to p-diazophenoxy hydroxypropyl cellulose or Alpha - or Beta -amylase chemically coupled to p-isothiocyanatophenoxy hydroxypropyl cellulose. 